Diagnostic Role of Salivary and GCF Nitrite, Nitrate and Nitric Oxide to Distinguish Healthy Periodontium from Gingivitis and Periodontitis.

Diagnosis of subclinical and early stage clinical periodontal dysfunction could prevent from further socioeconomic burden. The aim of this study was to assess the diagnostic applicability of nitric oxide and its end-metabolites in periodontal tissue health and disease. Forty-two patients were enrolled and divided into three groups according to gingivitis (GI) and clinical attachment level (CAL) indices: a healthy group (GI<1, CAL<1), b: gingivitis (GI>1, CAL>1) and c: periodontitis (CAL>1) with 14 patients in each group. Unstimulated saliva and gingival crevicular fluid (GCF) were collected. Samples were evaluated for nitrite, nitrate and total nitric oxide contents with the ELISA method. In addition, CAL, GI, plaque index (PI), decay, missing, filling (DMFT) and bleeding index (BI) scores were also recorded. Except for GCF nitrite content (P= 0.89), there was an increasing trend for measured biomarkers in both saliva and GCF (Periodontitis> gingivitis> healthy periodontium, P< 0.05). Data remained stable after simultaneous adjustment for DMFT and BI scores as confounding factors. Sensitivity, specificity, positive predictive value, negative predictive value, cut point and p- value were as the followings: GCF nitrate (0.71, 0.11, 0.29,0.43, 4.97, P= 0.04), nitric oxide GCF ( 0.64, 0.18, 0.28, 0.5, 10.12, P= 0.04), nitrite saliva (0.93, 0.96,0.93,0.96,123.48, P< 0.001), salivary nitrate (0.93, 0.96, 0.93, 0.96, 123.6, P< 0.001), salivary nitric oxide (0.93, 0.96, 0.93, 0.96, 246.65, P <0.001). Our results revealed that NO plays an important role in the process of destruction of periodontal tissues. Within the limitation of our study, detecting NO biomarker and its end metabolites in saliva is of more value to assess the periodontal health comparing to GCF.

introduced a cut-point system to distinguish healthy periodontium from periodontitis based on total salivary nitric oxide content (7). There is no published data about GCF nitric oxide content and its end metabolites (i.e., nitrate and nitrate) in normal periodontium, gingivitis and periodontitis.
The aim of the present study was to evaluate diagnostic power of GCF when compared to the whole saliva. Our hypothesis of superiority of GCF over saliva was based on mentioned premise that GCF is directly and solely affected by surroundingperiodontal tissue.

Study design
This case-control study was performed on patients who referred to the Department of

ELISA method
Two milliliters of unstimulated saliva were collected by spitting method as previously reported (7). The participants were asked to avoid eating, Then, nitrite is assayed using Griess reagent. This two-step assay method provides a simple and sensitive assay for monitoring nitric oxide production.

Statistics
Continuous data are presented in mean (±standard deviation). First, the data distribution was tested using the Kolmogorov-Smirnov test.

Ethical Approval
The present research was approved by Ethics Committee of Babol University of Medical Sciences and all researches undertook Helsinki treaty.   Figure 1 and Table 3.

Discussion
In this study, the diagnostic value of nitric oxide (NO) in saliva and GCF were examined to assess the periodontal health status. Results showed that the overall levels of nitric oxide, nitrite and nitrate were higher in saliva compared to GCF.
Also, the salivary nitric oxide increased in the order of healthy subjects, gingivitis patients and those suffering from periodontitis while GCF nitric oxide decreased in the same order.
To our knowledge, NO and its metabolites in saliva and GCF are being evaluated and compared among the three groups of healthy subjects, gingivitis and periodontitis patients for the first time. Unstimulated salivary samples were collected as the mastication affects it (11). The levels of NO and its metabolites were significantly different among three groups, except for GCF nitrite levels (P= 0.59), that even after adjustment with BI, differences did not reach a significant level (P = 0.15). All the above salivary levels were adjusted by DMFT index, BI and the GCF levels were adjusted by BI, PPD. It has been suggested that NO reflects the dynamic state of the patient to a static state. Thus, the pattern of disease progression in gingivitis patients with high BI (active) is different from that in periodontitis patients with high CAL and low BI (dormant advanced periodontitis). So all levels were moderated with BI as a key indicator of disease activity and affecting factor on the amount of NO and its metabolites. (11)(12)(13). Due to previously documented relationships between DMFT and the salivary NO levels as an antibacterial agent, the groups were moderated with the mean DMFT of their respective patients (5,(14)(15). Similarly, the reason for adjustment for PPD scores was that several researchers proved the relationship between NO and PPD (7,(16)(17).
The pathophysiological role of NO in periodontal disease was introduced in 2000 by Ozmeric et al. (18). It has been shown that bacterial lipopolysaccharide in the wall of periopathogenic bacteria causes death and apoptosis of periodontal ligament (PDL) through increased iNOS and phosphorylation of C-Jun N-terminal kinase (19)(20) al. (22)(23). It seems that these differences are due to the lack of moderation with BI as an effective factor and also DMFT in patients of various groups.
Inconsistencies among evaluated groups in various studies could also explain part of the differences.
GCF NO was reduced in order in the three studied groups and the GCF nitrate level was the highest in gingivitis group and the lowest in the periodontitis group. Ozer et al., in their study reported the greatest amount of NO in gingivitis patients (23). So according to their opinion, probably the secreted substances in periodontitis suppress the production of NO (23). Also, as a defence molecule, it may be consumed when disease progresses to combat the interfering oxidative and infectious process (3,24). This could be due to the fact that consumption of NO as an antibacterial agent is for controlling the bacteria that reduce its level (3).
In our research, the amount of total NO, nitrite and nitrate of saliva were found to be more patients, but no gingivitis group was enrolled (7).
The present investigation is limited in some aspects. Patients with advanced periodontitis were not included. A better cut point could be presented with higher sample size and including patients with CAL more than 5 mm. Another issue in this particular group is that as previously shown there is an exponential and not linear correlation with PPD and NO content. This fact may be related to more remarkable influence over the GCF ingredients of advanced periodontitis group with deeper PPD (7).
Our final number of enrolled patients was lesser than the primary design that had lessen the power of our analysis. As previously suggested, simultaneous measuring of NOS substrates such as available oxygen, arginine and reactive oxygen species (ROS) could reflect the competency of NOS systems more accurately (12). This could be accomplished by developing laboratory systems that could measure eNOS and iNOS within saliva and GCF eliminating the need for tissue sampling.
In conclusion, nitric oxide plays a role in the destruction of periodontal tissues. By considering the limitations of current study, the measured levels in the patients saliva compared with their GCF are of higher paraclinical and diagnostic value.
Nevertheless, more sensitive techniques with measurement of iso-enzymes of nitric oxide synthase may warrant more conclusive assumptions.